Hypoxic pulmonary vasoconstriction (HPV) is an important physiological process that maintains the sufficient matching of regional alveolar ventilation and pulmonary perfusion in the lungs, but may also lead to pulmonary hypertension. An increase in [Ca2+]i in pulmonary artery smooth muscle cells (PASMCs) is a key factor in HPV. The hypoxic increase of [Ca2+]i may occur due to intracellular Ca2+ release from the sarcoplasmic reticulum via ryanodine receptors/Ca2+ release channels (RyRs), which are activated by reactive oxygen species (ROS) signaling from mitochondria and NADPH oxidase (NOX). NOX-dependent hypoxic ROS generation is secondary to mitochondrial ROS. However, the molecular basis by which hypoxia generates ROS signaling still remains unclear. Here, we propose an innovative central hypothesis that rieske in the mitochondrial complex III is a key, initial hypoxic sensing molecule, which mediates the hypoxic ROS generation (in the complex III, mitochondria and then cytosol), RyR activation, [Ca2+]i increase and contraction in PASMCs, leading to pulmonary vasoconstriction and hypertension. In addition, rieske expression may vary to determine the heterogeneity of hypoxic responses in resistance and conduit pulmonary as well as systemic artery SMCs. To test this novel hypothesis, we will address the following fundamental questions (Specific Aims): (1) is rieske in the mitochondrial complex III a key, initial molecule that mediates the hypoxic ROS production, RyR activation, [Ca2+]i increase and contraction in PASMCs~ (2) are rieske-mediated hypoxic ROS signaling and attendant RyR-dependent Ca2+ signaling important for hypoxic pulmonary hypertension~ and (3) does rieske vary in expression to mediate the heterogeneity of hypoxic ROS signaling, RyR-mediated signaling in pulmonary and systemic artery SMCs? These specific aims will be implemented using complementary molecular, biochemical, physiological, and genetic approaches at the molecular, complex, mitochondrial, cellular, tissue and organism levels. The findings will extend our understanding of the mechanisms for hypoxic Ca2+ and contractile responses in PASMCs and also the heterogeneity of hypoxic responses in pulmonary and systemic artery SMCs. Our data may also help to identify novel therapeutic targets for pulmonary hypertension and other related lung diseases. PUBLIC HEALTH RELEVANCE: Hypoxia causes vasoconstriction in pulmonary arteries, which is an important physiological process to maintain the sufficient matching of regional alveolar ventilation and pulmonary perfusion in the lungs, but may also lead to pulmonary hypertension and even heart failure. However, the molecular mechanisms for hypoxic pulmonary vasoconstriction remain unclear. This application seeks to determine the potential role of rieske in the mitochondrial complex III as a key, initial molecule to mediate hypoxic reactive oxygen species, calcium and contractile responses leading to pulmonary vasoconstriction and hypertension. In addition, rieske expression may vary to mediate the heterogeneity of hypoxic cellular responses in pulmonary and systemic artery smooth muscle cells. The findings from the proposed studies in this application will greatly enhance our understanding of hypoxic cellular responses in vascular cells and may also help to identify novel, specific therapeutic targets for pulmonary hypertension and other relevant lung diseases.